Imidacloprid Induces Oxidative Stress and Genotoxicity in Nile Tilapia: The Role of Ascorbic Acid Combined Exposure

Imidacloprid (Imid), a systemic neonicotinoid insecticide, is broadly used worldwide. It is reported to contaminate aquatic systems. This study was proposed to evaluate oxidative stress and genotoxicity of Imid on Nile tilapia (Oreochromis niloticus) and the protective effect of ascorbic acid (Asc). O. niloticus juveniles (30.4 ± 9.3 g, 11.9 ± 1.3 cm) were divided into six groups (n=10/replicate). For 21 days, two groups were exposed to sub-lethal concentrations of Imid (8.75 ppm, 1/20 of 72h-LC 50 & 17.5 ppm, 1/10 of 72h-LC 50 ); other two groups were exposed to Asc (50 ppm) in combination with Imid (8.75 & 17.5 ppm); one group was exposed to Asc (50 ppm) in addition to a group of unexposed sh which served as controls. Oxidative stress was assessed in the liver where the level of enzymatic activities including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in addition to mRNA transcripts and, Lipid peroxidation (LPO) were evaluated. Moreover, mitotic index (MI) and comet assay were performed, in addition to the erythrocytic micronucleus (MN), and nuclear abnormalities (NA) were observed to assess genotoxicity in sh. Imid exposure induced signicant (p (cid:0) 0.05) changes in the antioxidant prole of the juveniles' liver by increasing the activities and gene expression of SOD, CAT and GPX as well as elevating the levels of LPO. DNA strand breaks in gill cells, erythrocytes and hepatocytes along with erythrocytic MN and NA were also signicantly elevated in Imid-exposed groups. MI showed a signicant (p (cid:0) 0.05) decrease associated with Imid exposure. Asc administration induced a signicant amelioration towards the Imid toxicity (8.75 & 17.5 ppm). A signicant protective potency against the genotoxic effects of Imid was evidenced in Asc co-treated groups. Collectively, results highlight the importance of Asc as a protective agent against Imid-induced oxidative stress and genotoxicity in O. niloticus juveniles.


Introduction
The aquatic environment is continually contaminated with agricultural chemicals, pesticides and urban activities. Aquatic pollutions affect the health and survival status of the organisms 1,2 .. In most tropical and subtropical regions, tilapia is introduced for sh farms and constitute an important dietary item for human consumption. Thus, tilapia became the most common freshwater shes in aquaculture worldwide 3,4 .
Neonicotinoids are one of the most used synthetic groups of insecticides owned to their high effectiveness against a wide range of insects. They are replacing older classes of insecticides such as carbamate and organophosphate worldwide because they are non-volatile, and easily soluble in water 5,6 .
Neonicotinoids are widely contaminating the environment due to their absorption by the seeds, and then their direct release through leaching, drainage, run-off, or snowmelt 7,8 . Imidacloprid (Imid) was the rst neonicotinoid introduced in 1991 and since has been one of the key ingredients of several pest control programs 9,10 . Xenobiotics or toxic chemicals including Imid may affect the endogenous and exogenous reactive oxygen species (ROS) balance and can subsequently suppress the antioxidant defenses or induce macromolecules oxidative damage in many organisms [11][12][13] . Imid causes cellular stress when consumed by fresh water Cyprinus carpio for 30 days leading to the decline of the population size in its natural habitat 2 . Its toxicity in Oncorhynchus mykiss and Danio rerio 14 , Cyprinus carpio 2 and O. niloticus 4 was reported. In carp sh, it induced severe in ammation, oxidative stress and histopathological lesions in the gills, liver, and brain 15 . Australoheros facetus, exposed acutely to environmentally relevant concentrations of Imid (1 to 1000 µg/L), showed oxidative damage affecting the genetic integrity of the sh 16 . Changes of the cellular detoxi cation and oxidative status of Corbicula uminea were also reported 17 . Fish leukogram was reported to be affected due to sub-lethal concentrations of Imid (140 and 280 mg/L of imidacloprid for 96 h) 4 .
Biological structures and functions can be early disturbed by DNA damage and micronucleus (MN) formation which could lead to a genotoxicity eventually associated with carcinogenicity and reproductive disorders [18][19][20] . Using the comet assay, a higher level of DNA damage was reported in shes due to genotoxicity [21][22][23] . In Prochilodus lineatus sh, MN and DNA damage were evidenced in the erythrocytes as a result of Imid exposure 6,24 . Vitamin C, chemically known as ascorbic acid (Asc), is well known for its strong antioxidant potency [25][26][27] . It is a good reducing agent and inhibits lipid peroxidation 28 , And even in small doses, its effectiveness for redox recycling was proved 29 . ROS conversion to harmless metabolites in addition to the protection and restoration of normal cellular metabolism and functions are mediated by endogenous enzymatic and non-enzymatic antioxidants 30 . Previously, it was reported that Asc is able to scavenge free radicals in pesticides-induced oxidative stress in various sh species 12,[31][32][33] . In earlier studies, bene cial effects of Asc on adult Oncorhynchus mykiss were proven 34 Thus, the purpose of the current study was to investigate the potential protective effect of Asc to overcome the hazards of insecticide applications in the surrounding environment. It was crucial to investigate the change of the oxidative markers including SOD, CAT, GPX and LPO in addition to evaluating the associated genotoxicity. O. niloticus is considered a rich dietary source and one main aspect of the shery future, thus concluding the Imid-induced oxidative stress and genotoxicity in its juveniles and the possible preventive strategies such as Asc administration in the aquaculture, is warranted. To the best of our knowledge, this is the rst report focusing on this point on O. niloticus.

Dose assessment in water
In order to assess the degradation of the tested compounds in the experimental water, HPLC was performed for Imid and Asc. Results after 24 h of compounds' application revealed that the degradation of Asc in water was approximately 30%. However, Imid has higher stability in water with a degradation around 9 %.
Lethal concentration, general conditions and health Imid concentrations of 17.5 ppm (1/10 of LC 50 ) and 8.75 ppm (1/20 of LC 50 ) used in this study were chosen based on the determined lethal concentration of Imid in O. niloticus juveniles (175.32 ppm) after 72h (Fig. 1). During the experiment, there was no mortality in the studied groups. The characteristics (chemical and physical) of the water over the experimental period remained stable including Imid and Asc concentrations. Behavioral and morphological observations were detected in Imid-treated groups (8. 75 & 17.5 ppm) such as darkness in sh color, erected ns and sluggish movement.

Antioxidant markers in sh livers
In order to assess the oxidative stress in sh, the enzymatic activities of SOD, CAT and GPX were investigated in liver tissues (Fig. 2). Results revealed that SOD, CAT and GPX were elevated signi cantly (P < 0.05) by ~ 52.5, 26.6 & 106.3% respectively, in 8.75 ppm of Imid-exposed group. While higher dose (17.5 ppm) increased the activities by ~ 20.6, 58.8 & 19.5% respectively, with a signi cant differences except for GPX activities that was non-signi cant, when compared with control. Moreover, LPO levels were signi cantly elevated by ~ 18, 42.8% among low and high concentrations of Imid. Asc co-treatments showed a signi cant decrease in SOD, CAT and GPX levels by ~ 11.5 & 24.4, 5% in low dose and by 13.9, 38.4 & 32.5% respectively, in higher dose of Imid co-treated group when compared with Imid-exposed group. The activities of SOD in higher co-treated group and GPX in lower one showed non-signi cant (P < 0.05) elevations, however, other co-treated groups showed a signi cant ameliorative effect of Asc towards the oxidative stress induced by Imid. Further, LPO levels were signi cantly (P < 0.05) decreased by ~ 20.9, 32.6% in low and high dose of Imid co-treated groups respectively, compared to Imid-exposed groups. Asc treatment showed a signi cant decrease by ~ 21.6% compared to control.
Relative gene expression of SOD, CAT and GPX Antioxidant genes expression for SOD, CAT and GPX were investigated in liver tissues (Fig. 3). A signi cant (p 0.05) up-regulation of relative mRNA by ~ 10.6, 0.5 & 3 fold respectively were observed in 8.75 ppm of Imid-exposed group. However, 17.5 ppm Imid-exposed sh showed a signi cant higher upregulation than the low Imid concentration by ~ 25 & 1.5 for SOD and CAT respectively. In contrast, GPX showed a non-signi cant elevation by ~ 0.33 folds. Relative to Imid-exposed group, Asc co-treatments showed a protective effects by down-regulating the SOD, CAT and GPX mRNA expression by 10. Evidences of micronuclei (MN) and erythrocytic nuclear abnormalities (ENA) The frequencies of MN and ENA were assessed in the treated and control sh to monitor the genotoxic effect of Imid exposure (8.75 & 17.5 ppm) and the ameliorative effect of Asc (Fig. 5). Fish exposed to Imid showed a signi cant (p 0.05) increase in MN and ENA by ~ 214.8 & 676.6% in Imid-exposed group to lower dose and in parallel, the higher one by ~ 879 & 248.2 % respectively. In respect to the Imidexposed groups, Asc co-treatments showed a signi cant (p 0.05) decrease in MN  compared to their Imid-exposed groups. While, Asc-treated group alone showed a signi cant elevation in MI by ~ 19.2% when compared to control group (Fig. 6).

Discussion
The antioxidative system plays an crucial role in repelling exogenous pollution, and other stimuli that induce the production of the superoxide anion, the intracellular parental form of reactive oxygen species (ROS), which is a highly active molecule and thereby causing various damages to cells and organisms 35  CAT are considered the primary antioxidant enzymes that contribute to the balance of free radicals in organisms and their activation is thus needed 35 . The activity of CAT was signi cantly enhanced in the sh digestive glands and gills following 30 days of Imid exposure 17 . In agreement, in this research, the SOD, CAT and GPX activities in the liver tissues were signi cantly increased in shes receiving Imid treatments, which may be due to the production of ROS 35 . GPX like CAT and SOD are considered as an oxidative stress indicator and has a vital role in the protection by normalizing the ROS levels [37][38][39][40] .
Similarly to the nding of this study, Vieira et al. 6 reported that, in gills, lower concentration of Imid caused a signi cant elevations in SOD and GPX activities which were subsequently declined in correspondence to the increase of Imid concentration. This may be due to the over accumulation of free radicals that, exceeded the antioxidant defense systems ability and the impact of Imid on the antioxidant balance, these nding are in agreement with Saddick et al. 41 .
Levels of LPO is correlated to the antioxidant status 42,43 and re ects the loss of membrane integrity 11 prior to the cellular damage. The alterations in the antioxidant enzymes (SOD and CAT) in the current study suggest a state of oxidative stress accompanied by an elevation of ROS levels, and con rmed by the detection of high LPO levels in the liver of O. niloticus exposed to Imid. The activities and mRNA transcripts levels of SOD, CAT and GPX were signi cantly increased in Imid-exposed groups. Equally important, the elevation of CAT and GPX is considered as oxidant stress indicator in tilapias 43 . The differences between SOD activity and transcripts could be explained by a responses delay at different levels, or by the impact of toxicants on transcriptional or translational mechanisms 44,45 .
The disturbance of biological structures and functions could be correlated to DNA damage leading to genotoxicity 18 . DNA damage in hepatocytes, erythrocytes and gill cells was reported in sh exposed to Imid 24 . This may be due to the formation of H 2 O 2 which is di cult to be eliminated leading to oxidative DNA damage specially in the diminished antioxidant enzymatic activities 36,46,47 . The damage was explained by the entrance of pyrethroids to the nucleus through cell membranes and its interaction with DNA leading to DNA unwinding and genetic material damages 48 . Moreover, DNA damage may be occurred due to interacting with generated oxygen radicals and the formation of DNA-protein or DNA-DNA crosslinks 49 . Our results revealed that sh exposed to Imid for 21 days exhibited DNA damage that was increased in gill cells, hepatocytes and erythrocytes.
In the present study, MN and ENA showed a signi cant increase in Imid-exposed sh suggesting dysfunction of mitotic spindle and/or breaks of DNA strands of the hematopoietic tissues 50,51 . Our results are supported by previous reports where MN and ENA were seen earlier in sh following the administration of Imid 16,24 . and the process was owned to the fact that Imid can affect the erythrocytic nuclear membrane leading to the DNA fragmentation, MN and ENA formation in a time and dose dependency 46 . The current results are in agreement with Iturburu et al. 16 . The results of mitotic index, known as a cell division marker 52 , showed a decrease in sh exposed to sub-lethal concentrations of Imid. The in vitro aneunogenic effects may lead to cellular imbalances and this phenomenon was documented earlier for Imid exposure 53 . The decreased MI in rats exposed to malathion pesticide were reported 52 . As observed in several animal models, the genotoxicity is suggested to be mediated by the generation of oxidative stress associated with diminished acetylcholinesterase and GPX activities in addition to elevated SOD and CAT activities [54][55][56][57][58] .
Asc, a water-soluble vitamin 59 , is a non-enzymatic antioxidant agents acting on both extracellular and intracellular uids and able to neutralize many radicals 60 . In this study, Asc co-treatments decreased the antioxidant enzymes and LPO in liver leading to ameliorative effect against Imid-induced oxidative stress. At the level of genotoxicity, Asc co-treatments decreased DNA damage, MN and ENA, however, the MI was elevated. The impact of Asc is owned to the decreased ROS and LPO 61 which improved the antioxidant status either by radical scavenging or elevating the antioxidant defense system leading to alleviation of oxidative stress that affect DNA and other macromolecules in sh 51,54 . Several studies indicated that Asc is an effective protective tool against the tissue damage and toxicity caused in various organisms by environmental pollutants such as toxicants, pesticides and insecticides 33,62,63 . Due to the production of free radicals or ROS and peroxidation of cell membrane lipids, these chemicals contribute to cell, tissue or even animal death. Asc either acts as a free radical scavenger 64 and also increases the innate immunity of sh 27,65 in addition to preventing the mitochondrial oxidative damage which consequently reduces the DNA and other macromolecules damage 66-68 .
In conclusion, the current study provides a new insight on the protective effect of ascorbic acid supplementation against the oxidative stress and associated genotoxic damage resulted by imidacloprid exposure. As the best of our knowledge, this is the rst report linked the Imid exposure with Ascameliorated effect in tilapias namely O. niloticus juveniles taking into account their economical importance and the quality and safety measures of human consumption.

Animals
Animal management procedures were undertaken in accordance with the requirements of the Institutional Animal Care and Use Committee (IACUC), Menou a University, Egypt. The protocol of this study has been approved by the ethics review board of the IACUC of Faculty of Science (ID: MUFS-F-EC-1-20  50 ) and a concentration of 50 ppm for Asc as described by Ghazanfar et al. 33 . Fish were randomly divided into six groups (n = 10 sh per group, ve of them were processed for chromosomal preparation and the remained ve sh were used for other investigations). Fish were maintained in glass aquaria containing dechlorinated tap water (50 L). Frist group was used as control and the second group was exposed to Asc only. Other two groups were subjected to Imid concentrations of 17.5 ppm (1/10 of LC 50 ) and 8.75 ppm (1/20 of LC 50 ). Finally, two co-treatment groups were exposed to Imid and Asc (17.5 ppm -Imid + 50 ppm Asc) and (8.75 ppm-Imid + 50 ppm Asc). The experiment was performed for 21 days according to Al-Anazi et al. 27,33 , under static conditions. Water renewal (30%) was done daily to overcome the daily degradation of Imid and Asc as previously described 33,70 and in agreement with the HPLC results.. Further, all aquaria were laterally covered with black sheets to minimize the effect of light on Imid and Asc, where, Asc is light sensitive 71 . During the experiments, temperature measurements, dissolved oxygen, ammonia levels, pH and water conductivity were adjusted as acclimatization conditions.
After the exposure period, caudal vein puncture was used to collect blood samples and processed for MN test and comet assay. After blood sampling sh were sacri ced on ice immediately by medullar sectioning for liver removal. Organs were quickly stored in -20 ºC for gene expression and biochemical analyses. In addition, samples of liver and gills were freshly processed for comet assay.
For biochemical analyses, samples of liver tissues were homogenized (1:10 wt./v) in a 0.1M phosphate buffer solution (pH 7.1 containing 1mM Mercaptoethanol and 2mM EDTA). Samples were centrifuged (15,000 xg, 20 min, 4 ºC) and the supernatants were stored at -80 ºC for subsequent biochemical analyses. For all biochemical biomarkers assessment, the determination of liver protein content was done 72 . Experiments were done in triplicates.

Assessment of Imid and Asc degradation in water
HPLC quanti cation of Imid and Asc in water samples was done using High performance liquid chromatography (HPLC) analysis. After 24h of exposure period, water samples were collected in clean amber glass bottles and HPLC analysis was performed. The ZORBAX Eclipsed XDB-C18 column (4.6x 150 mm, 5um) and Zorbax C8 column (4.6 mm x 150 mm i.d., 5 µm) were used for chromatographic separation of Imid and Asc, respectively. The mobile phase consisted of methanol: water (60: 40%, respectively) for Imid and 0.01% tri uoroacetic acid in water and methanol (70: 30%, respectively) for Asc.
Identi cation and quanti cation of Imid was performed by HPLC-DAD using an Agilent 1260 device (Agilent Technologies, CA, USA).

Biochemical analyses
The homogenized liver supernatants were used to determine the lipid peroxidation as malondialdehyde (MDA) levels 73 . Results were presented in nmol/mg protein. The activities of superoxide dismutase (SOD) 74 , catalase (CAT) 75

Gene expression
Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to evaluate the expression of liver SOD, CAT and GPX genes in tilapias. Used primer sequences were illustrated in Table (1). The total RNA was extracted from tissue using the RNeasy Kit (Qiagen, Hilden, Germany) following the company's protocol. The Reverse Transcript kit (Qiagen, Hilden, Germany) was used cDNA synthesis. The qPCR of the β actin (a housekeeping gene) and studied genes were performed using a Qiagen QuantiTect SYBR Green PCR kit in a Rotor-Gene Q cycler (Qiagen, Hilden, Germany).

Chromosomes preparation
Chromosomal preparation was performed from tilapia Kidneys and the mitotic index was calculated 79,80 . Brie y, a volume of 1 mL / 100 g b.wt. of colchicine (0.05%) was injected into the abdominal cavity of sh two hours prior to the dissection. After the kidney removal, it was cut into small pieces before mixing with 5 mL of the hypotonic solution (0.075M KCl). All large pieces of the kidney tissues were discarded. At room temperature, the suspended cells was incubated for 20 minutes, and centrifuged for 5 min at 400 g. Dropwisely, cells were applied to the xation step using 5 mL of fresh cold xative (3 methanol: 1 acetic acid) before centrifugation. The xation process was repeated until the supernatant was cleared. A concentrated volume of each tube was dropped 15 cm high on a clean and 70% cold ethanol-dipped glass slide and left to dry at room temperature. The slide was conventionally stained for 30 minutes with 20% Giemsa solution, pH 6.8. Metaphases and prophases were evaluated over 1000 nuclei per slide to calculate the mitotic index.

Statistical analysis
After data normality checking (Shapiro Wilk test) and homoscedasticity (Levene's test) mean values of all groups were cross-compared using parametric (ANOVA) by multiple comparison post hoc test, Dunnett's test. Also, the signi cance between Insecticide-treated group and the corresponding co-treatment group was compared by independent t test. The data shown in the graphs were represented as means ± SD. The signi cant level of differences was considered at P < 0.05. Statistical analyses were done using the IBM SPSS software version 21.1 (New York, NY, USA).

Declarations Data availability
All data of this study are introduced in this published article. 79. Chen, T. & Ebeling A. Karyological evidence of female heterogamety in the mosquito sh, Gambusia a nis . Copeia, 1, 70-75 (1968 Figure 1 The dose respose of O. niloticus exposed to diferent concentrations (0 -350 ppm) of Imid (n= 10 sh).

Figures
Values were expressed as mean± standard deviation (SD), bars refer to SD.

Figure 6
The protective effect of ascorbic acid (Asc, 50 ppm) on the mitotic index changes induced by imidacloprid (Imid) (8.75 & 17.5 ppm) applied for 21 days in O. niloticus. A, representative Representative micro-photographs of studied groups, B, the mean values (M±SD). Values were expressed as mean ± standard deviation (SD), bars refer to SD, (n=5 sh), a indicates signi cant difference compared to control group (p 0.05), *, # indicates signi cant difference between co-treatment and corresponding Imidexposed (17.5 & 8.75 ppm) groups respectively (p 0.05).